The invention relates to novel uses of fucans in the context of repairing connective tissue lesions, and in particular for regulating fibroblast functions.
Fibroblasts play an essential role in the equilibrium and repair of connective tissues. They are in particular responsible for renewing extracellular matrix, and in return their functions are modified by the substances present in this matrix.
In particular, in the process of tissue remodelling and healing which intervenes after an injury, the connective tissue is the context for constant exchanges between all the cells involved in this process. These exchanges take place in particular via cytokines or soluble mediators transmitted by the extracellular matrix.
For example, in the covering connective tissues such as the gum and cutaneous tissues, the healing process begins, after the formation of a provisional matrix (red thrombus), with the recruitment of inflammatory cells (leukocytes, macrophages and polynuclear cells), which initiate a phase of destruction of the lesioned tissue.
These inflammatory cells participate in the destruction:
by secreting matrix proteases such as collagenase (MMP8), leukocytic elastase or cathepsin G),
by liberating cytokines, and in particular interleukin 1 (IL1), which stimulate the proliferation and migration of fibroblasts and of epithelial cells, and the expression, by these cells, of certain metalloproteases such as interstitial collagenase (MMP1) or gelatinase B (MMP9).
This destruction phase, which begins very soon after the injury, ends when the epithelium and its basement membrane have been reconstituted.
It is prolonged by repair and resolution phases in which the fibroblasts reconstruct and reorganize the collagen framework; the expression by the fibroblasts of gelatinase A (MMP2) is in particular observed, matrix metalloprotease actively participating in all the tissue remodelling phenomena.
Some pathologies are accompanied by a chronic inflammatory state of the connective tissue, in which the balance between the destruction, repair and resolution phases is upset, which leads to defective reconstruction of the lesioned tissue.
This phenomenon is in particular observed in the case of periodontal diseases, or periodontopathies.
The periodontium represents the set of structures (gum, dento-alveolar ligament, alveolar bone and cement) which provides the support for the tooth in its dental alveolus.
Periodontopathies reveal themselves by more or less localized, often recurring, inflammatory episodes of infectious origin at the end of which the periodontal tissue is not correctly reconstructed.
In this pathology, initiation of the inflammatory response and cell proliferation in response to the infectious lesion take place more or less normally. Conversely, the resolution phase is rarely satisfactory; at best repair of the destroyed tissue is observed, but not its complete regeneration, and each episode of the disease induces a tissue loss.
Untreated periodontopathies evolve towards the mobility and then the loss of teeth in adults over forty years of age. Since these periodontal diseases, in a more or less widespread form, concern approximately 10 to 15% of the population, they have considerable consequences in terms of public health.
Currently, most of the treatments proposed are directed towards mechanically unbinding the lesion and reducing the microbial attack by administering antiseptics or antibiotics.
Another therapeutic approach would consist in improving the quality of the repair process to allow it to result in complete regeneration. However, this approach in particular requires being able to stimulate, at the desired moment, the appropriate cell population so as to orient cell proliferations and thus control the processes of tissue modification.
With this aim, the inventors have studied the action of various polysaccharides; it is known that some of these molecules, such as glycosaminoglycans, participate in the composition of the proteoglycans present at the cell/extracellular matrix interface, and play a role in regulating cell functions. It is also known that glycosaminoglycans in a soluble form, for example heparin or dextran derivatives, can modify cell functions via their interaction with various components of the extracellular matrix.
For example, in the case of heparin, a stimulatory effect on cell proliferation was shown in the case of hamster lung fibroblasts, bovine lens epithelial cells [Ulrich et al., Biochem. Biophys. Res. Commun., 139, p. 728-732, (1986)] and capillary endothelial cells [Sudlalter et al., J. Biol. Chem., 264, p. 6892-6897, (1989)].
Conversely, it has also been observed that heparin inhibits the proliferation of certain cell types in a dose-dependent manner; this antiproliferative effect has principally been studied in the case of smooth muscle cells (SMC), for which the inhibition becomes apparent for concentrations of 1 xcexcg/ml of heparin in the culture media. Heparin opposes both the migration and the proliferation of the SMCs, but does not affect the re-endothelialization or the volume of the connective tissue [Clowes and Clowes, Lab. Invest, 52, p. 611-615, (1985); Clowes and Clowes, Circ. Res., 58, p. 839-845, (1986); Clowes et al., J. Cell. Biol., 107, p. 1939-1945, (1988)].
An inhibition of proliferation has also been observed for other cell types, such as for example sclera fibroblasts [Del Vecchio et al., Invest. Ophtalmol. Vis. Sci., 29, p. 1272-1276, (1988)], 3T3 fibroblasts (mouse embryo fibroblasts which conserve contact inhibition) [Paul et al., Thromb. Res., 18, p. 883-888, (1980)], rat cervix epithelial cells [Lyons-Gioradano et al., Biochem. Biophys. Res. Commun., 148, p. 1264-1269, (1987)] and human dermal fibroblasts.
Ferrao and Mason [Biochem. Biophys. Acta. 1180, 225-230, (1993)] have studied the action of various polysaccharides on human dermal fibroblast proliferation, and indicate that at concentrations of about 100 xcexcg/ml, heparin, heparan sulphate, pentosan polysulphate and a fucoidan inhibit this proliferation, whereas chondroitin sulphate, dermatan sulphate and hyaluronate have no effect. It is indicated that the inhibitory effect on proliferation leads to a stimulation of type I collagen synthesis. Conversely, an inhibition of collagen I synthesis is observed when the polysaccharides are added to cultures which have reached confluence.
It thus appears that the action of polysaccharides on cell functions is complex and can vary according to the polysaccharide, cell type and tissue concerned, as well as according to the polysaccharide concentration used and the state of the cells.
In the context of research directed towards elucidating the mechanism of action of various polysaccharides on fibroblast functions, and in particular those involved in tissue regeneration, the inventors were in particular interested in fucans.
Fucans are sulphated polysaccharides which participate in the constitution of the cell walls of shoots of brown algae (Pheophyceae); they are also present in some marine animals, such as sea urchins and sea cucumbers. Raw fucan, also termed fucoidan, obtained by acid extraction from the cell walls of brown algae shoots, consists of a heterogeneous population of molecules which comprises principally sulphated L-fucose polymers of high average molar mass (100,000 to 800,000 g/mol).
Fucans have varied biological activities: it has thus been shown that they possess anticoagulant, antithrombotic [T. Nishino and T. Nagumo, Carbohydr. Res. 229, p. 355-362, (1992); Application EP 0403 377; S. Colliec et al. Thromb. Res. 64, p. 143-154 (1991); S. Soeda et al. Thromb. Res. 72, p. 247-256 (1993); Mauray et al. Thromb. Haemost. (5) 1280-1285 (1995)], antiviral [M. Baba et al. J. AIDS, 3, p. 493-499, (1990)], antiangiogenic [R. Hahnenberger and A. M. Jackobson, Glycoconjugate J., 8, 350-353 (1991)] and anticomplementary [C. Blondin et al., Mol. Immunol., 31, p. 247-253, (1994)] activities. It has also been observed that they can act as modulators of cell adhesion [C. G. Glabe et al., J. Cell Sci., 61, p. 475-490, (1983)], of growth factor release [D. A. Belfort et al., J. Cell. Physiol. 157, p. 184-189, (1993)], of proliferation of tumour cells [M. Ellouali et al., Anticancer Res., 13, p. 2011-2020 (1993); D. R. Coombe et al., Int. J. Cancer, 39, pp. 82-90, (1987); D. Riou et al., Anticancer Res., 16, 1213-1218 (1996)] and of vascular smooth muscle cells [Logeart et al., Eur. J. Cell. Biol., 74, pp. 376-384 (1997)], and can block spermatozoid/ovule interactions in various species [M. C. Mahony et al., Contraception, 48, p. 277-289, (1993)].
Preparations of fucans of average molar mass lower than 20,000, or even than 10,000 g/mol, which facilitates their use in a therapeutic context, were obtained, for example, by controlled acid hydrolysis of fucan of high molar mass (Patent EP 0,403,377 in the name of IFREMER), or by radical depolymerization (Application PCT WO/9708206 in the name of IFREMER and CNRS).
In the report which will follow, the term: xe2x80x9cfucanxe2x80x9d encompasses both the fucans of high molar mass and the preparations of lower molar mass obtained from them.
The inventors have observed that fucans possess a profile of activity on fibroblast functions which is different from that of heparin. They have in particular observed, by testing these two polysaccharides under the same conditions, that whereas heparin inhibits both dermal fibroblast proliferation and that of gum fibroblasts, fucans activate dermal fibroblast proliferation while at the same time inhibiting that of gum fibroblasts. In addition, the inventors have also observed that fucans modify the morphology of dermal fibroblasts which round off, whereas gum fibroblasts, on the contrary, conserve a fibroblastic morphotype.
The inventors have also observed that fucans increase the amount of proteins in the cell layer and the activity of MMP2 (gelatinase A), and inhibit leukocytic elastase.
These effects reveal themselves both on dermal fibroblasts and on gum fibroblasts.
A subject of the present invention is the use of a fucan for obtaining a medicinal product which modifies the expression and/or the activity of fibro-blastic metalloproteinases, and in particular of MMP2, and which inhibits leukocytic elastase.
Fucans thus make it possible to control proteolytic activity in connective tissues such as dermal and gum tissues, and in particular to limit elastase activity, which exhibits considerable destructive potential with respect to connective macromolecular structures, while at the same time on the contrary promoting the activity of proteases which participate in tissue reconstruction, such as MMP2.
According to a preferred embodiment of the present invention, said medicinal product can also be used to inhibit the proliferation of gum fibroblasts and to activate their collagen synthesis. It allows the treatment of periodontal pathologies via an improvement in the resolution phase.
Specifically, due to the combination of a regulation of proteolytic activities (in particular activation of MMP2 and inhibition of elastase) with an inhibition of gum fibroblast proliferation, an increase in the synthesis of a physiological matrix, and the conservation of the fibroblastic morphotype, fucans make it possible to engage these gum fibroblasts in the remodelling pathway which is required for any process of tissue repair or regeneration.
According to another embodiment of the present invention, said medicinal product can also be used to activate the proliferation of dermal fibroblasts and their collagen synthesis. It thus allows the treatment of dermal lesions by improving the recovery phase of the lesioned tissue.
The medicinal products obtained in accordance with the invention can be administered generally (orally or parenterally). They can also be administered locally, in the form of gels, creams, ointments, lotions, lozenges, mouthwashes, etc.
They can also be administered in situ via substrates, resorbable or nonresorbable devices such as for example delayed-release supports, or slowly-disintegrating sponges.
Fucans can also be used in cosmetology, as fibroblast proliferation activators in the context of treatments aimed at aesthetics, for example of antiwrinkle treatments or of prevention of skin ageing, etc.
The present invention will be better understood with the aid of the further description which will follow, which refers to examples demonstrating the activity of fucans on dermal and gum fibroblasts.